1. Field of the Invention
This invention relates to diagnostic products and methods which utilize iodine-starch indicator systems in assays for reaction products from beta-lactamase catalyzed penicillin or penicillin-like substrates.
2. Prior Art
The penicillins and cephamycins have proven to be among the most preferred and generally useful antibiotics in the medical arsenal. It is of considerable conern, therefore, that recently several bacteriological species hitherto usefully and indeed preferably treated by administration of penicillins or cephamycins have been found to display decreased susceptibility to these antibiotics. A certain portion of such resistant bacteriological species have been shown to produce the enzyme beta-lactamase (at times referred to as penicillinase). For example, resistance to ampicillin by Haemophilus influenzae is reported and discussed by Thornsberry et.al. in Antimicrobial Agents and Chemotherapy, Vol. 6 No. 5 pages 653-654 (1974). Haemophilus influenzae causes acute bacterial meningitis of particular virulence in children. Another bacteria, Neisseria gonorrhoreae, has also been found to have penicillin resistant strains.
The penicillins and cephamycins probably inhibit bacteria by preventing cross-linking of the peptide chains necessary in formation of the bacterial cell walls. Both the penicillin and cephamycins include a 4-membered ring, denominated as the beta-lactam ring, which contains peptide bond as the functional site for the inhibitory function of penicillins and cephamycins. When bacteria are inhibited in their cell-wall synthesis, they are vulnerable to adverse environmental conditions or their own autolytic enzymes. When bacteriological species produce beta-lactamase, the beta-lactamase catalyzes a reaction in which the beta-lactam ring is ruptured. Hydrolysis results which causes destruction of the inhibitory capability of the penicillins and cephamycin substrates.
When such substrates have undergone beta-lactam ring cleavage (rupture of the peptide bond) the resulting structure is hereinafter throughout referred to for convenience as a penicilloic acid type moiety. Penicilloic acid type moieties contain a non-acylated amino group which is capable of reducing iodine to iodide.
Recently, the prior art has described B-lactamase production assay techniques which depend upon the interaction of a localized reductive reaction of iodine to iodide by penicilloic acid type moieties with a starch-iodine indicator system.
Among these reports, Jorgensen et.al. Antiomicrobial Agents in Chemotherapy, Vol. 11, No: 2, pages 1087-1088 (1977) discloses paper strips which have been impregnated with starch and potassium penicillin G as substrate. The impregnation is by immersion of the paper strips in a 0.2 wt.% starch and 1 wt.% penicillin solution. These strips are then moistened with either Gram's iodine (0.013 M iodine) or Lugol's iodine (as usually used, 0.039 M iodine). When colonies of bacteria are applied, a pale discolorization in the area of application provides a presumption of beta-lactamase production (iodine reduction to iodide).
Another literature report, Odugbemi et.al., British Medical Journal, pg. 500 (1977), teaches that various paper strips of unknown starch concentrations are soaked in a phosphate-buffered saline solution containing 10 wt.% of benzyl penicillin substrates. Bacterial colonies are then applied to the paper strips, incubated and then flooded with Gram's iodine diluted 1 to 2 (0.007 M iodine). A pale discolorization, read five minutes after iodine flooding, provides a presumption of beta-lactamase.
Neither the Jorgensen nor the Odugbemi paper strips and techniques have been found to provide satisfactory assay results. These prior products and techniques are not sufficiently sensitive or reproducible for large scale, hospital screening and/or public health use.
The Jorgensen teachings have been found deficient primarily in two respects. First, the low concentration of substrate taught by Jorgensen is believed to result in unfavorable kinetics for enzyme activity so that maximum reactive velocity is not attained, or the actual reaction velocity is not a direct function of the enzyme concentration. Second, the undiluted use of Gram's, and particularly the use of Lugol's, iodine presents problems in that development of decolorization in the area of bacteria application is frequently masked by excess iodine (in relation to the amount of penicilloic acid type moiety produced in a reasonable period of time). Thus, despite the application of known beta-lactamase producing bacteriological strains (as confirmed by other methods), the Jorgensen paper strip and technique frequently results in no discernible fading, overly slow, or only slight fading in the area of bacteria application.
The Odugbemi teachings also have been found unsatisfactory in several aspects. First, colonies of the bacteriological species must be incubated upon the paper strips in the presence of substrate. In large scale screening, such incubation is not convenient. Second, dilution of the Gram's or Lugol's iodine solution is inconvenient, since these solutions are stock concentrations for a multitude of purposes, and, more importantly, dilution results in a background color which is too pale for sensitive contrast with the area of bacteria application. Also, the lack of control over starch concentrations of the Odugbemi strips creates difficulty in reproducibility.
The present invention is directed to overcome one or more of the problems of the prior art assay products and techniques.